Project 1: Testing the mechanism by which vinculin, talin, actin and integrin mediate the dynamic, force transducing linkage between actin and the ECM in FAs. [unreadable] [unreadable] Personnell: Ingo Thievessen[unreadable] [unreadable] The goal of this project is to dissect the molecular mechanism of talin and vinculins role in mediating the tunable force-transducing molecular clutch linkage between actomyosin contraction and integrins in FA of migrating fibroblasts. This includes determining what molecular functions of talin and vinculin are responsible for their fascinating kinematic coupling to actin retrograde flow that we discovered by correlational qFSM, and secondly to understand how the molecular functions, dynamics, and force transmission are related. Based on the literature, our hypothesis is that talin is the primary mediator of the force-transmitting link between actin and integrin, and the strength of this link is reinforced by a talin-vinculin-actin interaction. We suspect that the kinematic coupling of talin to actin motion we see requires its direct actin-binding and/or an interaction with vinculin, while the vinculin-actin kinematic coupling requires vinculin activation and actin binding. We suspect that complete talin and vinculin coupling to actin retrograde flow corresponds to a low force transmitting state of the FA. In contrast, immobilization of talin in the FA requires its binding to integrin, and its concurrent interaction with actin results in intermediate force transmission, while its additional binding and activation of vinculin is required for high force transmission.[unreadable] In this project, we will test our hypothesis by first analyzing cell migration, actin dynamics, actin-FA molecule kinematic coupling and force transmission in migrating fibroblasts lacking vinculin or talin in our FSM and force measurement assays and analyze the data using qFSM, correlational qFSM and Cytoprobe software developed by the Danuser Lab. We will then focus on well-characterized talin and vinculin mutants deficient in specific binding activities added-back to fibroblasts lacking these proteins and apply this assay and analysis scheme to probe the molecular mechanism of force transmission from the actin cytoskeleton to the extracellular environment.[unreadable] [unreadable] Ingo has been able to derive a mouse in collaboration with Robert Ross, (Cardiology, UCSD) that has a cardiac-specific cre-inducible knockout of the single vinculin gene to sereve as a source of endothelial cells for this study. He has additionally developed cell isolation and imaging capabilities that allow performing the experiments. Image analysis is done in collaboration with Gaudenz Danuser, Scripps. Progress is ongoing.[unreadable] [unreadable] Project 2: Three dimensional superresolution fluorescence microscopy reveals protein stratification in focal adhesions[unreadable] [unreadable] Personnell: Pakorn Kanchanawong[unreadable] [unreadable] Focal Adhesions (FA) are dynamic structures consisting of large numbers (>150) of different proteins that mechanically link the actin cytoskeleton to the extracellular matrix (ECM). Despite the central role of FA in cell migration and the wealth of biochemical and cell biological data on FA proteins, it remains virtually unknown how these proteins are organized within FA. Based on the differential dynamics of distinct FA proteins we previously observed using fluorescent speckle microscopy, we hypothesized that FA proteins may be organized into stratified layers within FA that serve as dissipative elements in a molecular clutch to form a regulatable, force-transducing link between the actin cytoskeleton and the ECM. To test this hypothesis, we employed a 3-dimensional superresolution fluorescence microscopy technique, interferometric photoactivated localization microscopy (iPALM), to determine sub-20 nm z-axis localizations of several key structural components of FA labeled with photoactivatable fluorescent proteins and expressed in U2OS cells plated on a fibronectin-coated substrate. Farnesylated tdEosFP was used to demarcate the cytoplasmic face of the plasma membrane and was localized at 20 nm above the substrate. Paxillin, talin, and vinculin were distinguished by distinct vertical distributions, with the highest densities at 25, 30, and 50 nm above the substrate plane, respectively. Paxillin and talin formed narrow distributions with FWHM <15 nm, while the vinculin layer was broader with FWHM 25 nm. Actin appeared to be largely excluded from the bottom layers of FA up to a height of 50 nm, with the highest density observed at 90 nm above the substrate plane. iPALM reveals for the first time that different FA proteins are segregated into distinct layers parallel to the substrate plane. The protein stratifications in FA provide a structural context for the mechanosensing and mechanotransducing functions of FA.[unreadable] [unreadable] The technique of iPALM microscopy is performed in collaboration with Harald Hess, Eric Betzig, and Gleb Stengle (Janelia), Michael Davidson (FSU), Cathy and Jim Galbraith (NIDCR and NINDS), and Jennifer Lippincott Schwartz and Jennifer Gilette (NICHD) and a manuscript describing this method has been submitted. The results of the studies described above will be presented as a poster at the American Society for Cell Biology meeting in December 2008.[unreadable] [unreadable] Project 3: Actin dynamics correlate with force transmission through focal adhesions[unreadable] [unreadable] Personell: Clare Waterman[unreadable] The ability of cells to spatially and temporally regulate traction forces[unreadable] on their extracellular matrix is fundamental to tissue morphogenesis and[unreadable] directed cell migration. Forces generated in the actin filament (F-actin)[unreadable] cytoskeleton are transmitted through the cell plasma membrane to the[unreadable] extracellular matrix via mechano-sensitive focal adhesions1. In migrating[unreadable] cells, F-actin and focal adhesions exhibit stereotypical patterns of[unreadable] assembly, disassembly and motion2-13. It is well appreciated that an intact[unreadable] F-actin cytoskeleton is required for cellular force generation; however, the[unreadable] role of F-actin motion dynamics in force generation is unknown. We show[unreadable] here that F-actin motion spatially correlates with traction stresses on the[unreadable] extracellular matrix. Near the cell edge, traction stress and F-actin speed[unreadable] are inversely correlated, suggesting that focal adhesions strengthen by[unreadable] slowing F-actin and engaging it to the stationary extracellular matrix.[unreadable] However, instead of observing maximal traction stress when F-actin motion[unreadable] is minimized, we find that an intermediate speed of F-actin motion marks a[unreadable] switch to focal adhesion weakening. The switch from focal adhesion[unreadable] strengthening to weakening is not correlated with focal adhesion protein[unreadable] density, age, stress magnitude, assembly/disassembly status or[unreadable] subcellular position. In contrast, the F-actin speed associated with maximal[unreadable] traction stress and the transition to adhesion weakening is strikingly[unreadable] robust. Thus, we suggest F-actin motion dynamics may be an important[unreadable] regulator offocal adhesion-mediated traction forces at the leading edge of[unreadable] migrating cells.[unreadable] [unreadable] To achive this, we developed high resolution traction microscopy in collaboration with Ulrich Schwartz and Benedikt Sabass. [unreadable] [unreadable] This work has been performed in collaboration with Margaret Gardel (University of Chicago), Gaudenz Danuser and Lin Ji(Scripps), Ulrich Schwartz and Benedikt Sabass (Heidelberg University) and has been presented at mutliple meetings and symposia and has been submitted for publication.